The present application concerns micromachined or microfabricated devices known as xe2x80x9cbiochipsxe2x80x9d and more particularly biochips employing magnetic forces and methods of utilizing such biochips for performing chemical, biological and biochemical reactions and assays.
As a novel and emerging technology in life science and biomedical research during last several years, biochip technology can be applied to many areas of biology, biotechnology and biomedicine including point-mutation detection, DNA sequencing, gene expression, drug screening and clinical diagnosis. Biochips refer to miniaturized devices having characteristic dimensions in the micrometer to millimeter range that can be used for performing chemical and biochemical reactions. Biochips are produced using microelectronic and microfabrication techniques as used in semiconductor industry or other similar techniques, and can be used to integrate and shrink the currently discrete chemical or biochemical analytical processes and devices into microchip-based apparatus. Recent scientific literature shows a plethora of uses for these devices.
The reader""s attention is drawn to the following articles for an appreciation of the breadth of biochip uses. xe2x80x9cRapid determination of single base mismatch mutations in DNA hybrids by direct electric field controlxe2x80x9d by Sosnowski, R. G. et al. (Proc. Natl. Acad. Sci., USA, 94:1119-1123 (1997)) and xe2x80x9cLarge-scale identification, mapping and genotyping of single-nucleotide polymorphisms in the human genomexe2x80x9d by Wang, D. G. et al. (Science, 280: 1077-1082 (1998)) show current biochip use in detection of point mutations. xe2x80x9cAccurate sequencing by hybridization for DNA diagnostics and individual genomics.xe2x80x9d by Drmanac, S. et al. (Nature Biotechnol. 16: 54-58 (1998)), xe2x80x9cQuantitative phenotypic analysis of yeast deletion mutants using a highly parallel molecular bar-coding strategyxe2x80x9d by Shoemaker, D. D. et al. (Nature Genet., 14:450-456 (1996)), and xe2x80x9cAccessing genetic information with high density DNA arrays.xe2x80x9d by Chee, M et al., (Science, 274:610-614 (1996)) show biochip technology used for DNA sequencing. The use of biochip technology to monitor gene expression is shown in xe2x80x9cGenome-wide expression monitoring in Saccharomyces cerevisiaexe2x80x9d by Wodicka, L. et al. (Nature Biotechnol. 15:1359-1367 (1997)), xe2x80x9cGenomics and human diseasexe2x80x94variations on variation,xe2x80x9d by Brown, P. O. and Hartwell, L. and xe2x80x9cTowards Arabidopsis genome analysis: monitoring expression profiles of 1400 genes using cDNA microarrays.xe2x80x9d by Ruan, Y. et al. (The Plant Journal 15:821-833 (1998)). The use of biochips in drug screening is illustrated in xe2x80x9cSelecting effective antisense reagents on combinatorial oligonucleotide arrays.xe2x80x9d by Milner, N. et al. (Nature Biotechnol., 15:537-541 (1997)), and xe2x80x9cDrug target validation and identification of secondary drug target effects using DNA microarray.xe2x80x9d by Marton, M. J. et al. (Nature Medicine, 4:1293-1301 (1998)). Examples of clinical diagnostic use of biochips are illustrated in xe2x80x9cCystic fibrosis mutation detection by hybridization to light-generated DNA probe arrays.xe2x80x9d by Cronin, M. T. et al. (Human Mutation, 7:244-255 (1996)), and xe2x80x9cPolypyrrole DNA chip on a silicon device: Example of hepatitis C virus genotyping.xe2x80x9d by Livache, T. et al. (Anal. Biochem. 255:188-194 (1998)). These references are intended to give a notion of the wide range of biochip uses.
A variety of biochips have biomolecules (e.g., oligonucleotides, cDNA and antibodies) immobilized on their surfaces. There are a number of different approaches to make such chips. For example, the light-directed chemical synthesis process developed by Affymetrix (see, U.S. Pat. Nos. 5,445,934 and 5,856,174) is a method of synthesizing biomolecules on chip surfaces by combining solid-phase photochemical synthesis with photolithographic fabrication techniques. The chemical deposition approach developed by Incyte Pharmaceutical uses pre-synthesized cDNA probes for directed deposition onto chip surfaces (see, e.g., U.S. Pat. No. 5,874,554). The contact-print method developed by Stanford University uses high-speed, high-precision robot-arms to move and control a liquid-dispensing head for directed cDNA deposition and printing onto chip surfaces (see, Schena, M. et al. Science 270:467-70 (1995)). The University of Washington at Seattle has developed a single-nucleotide probe synthesis method using four piezoelectric deposition heads, which are loaded separately with four types of nucleotide molecules to achieve required deposition of nucleotides and simultaneous synthesis on chip surfaces (see, Blanchard, A. P. et al. Biosensors and Bioelectronics 11:687-90 (1996)). Hyseq, Inc. has developed passive membrane devices for sequencing genomes (see, U.S. Pat. No. 5,202,231).
There are two basic types of biochips, i.e., passive and active. Passive biochips refer to those on which chemical or biochemical reactions are dependent on passive diffusion of sample molecules. In active biochips reactants are actively moved or concentrated by externally applied forces so that reactions are dependant not only on simple diffusion but also on the applied forces. The majority of the available biochips, e.g., oligonucleotide-based DNA chips from Affymetrix and cDNA-based biochips from Incyte Pharmaceuticals, belongs to the passive type. There are structural similarities between active and passive biochips. Both types of biochips employ of arrays of different immobilized ligands or ligand molecules. Herein, xe2x80x9cligands or ligand moleculesxe2x80x9d refers to bio/chemical molecules with which other molecules can react. For instance, a ligand may be a single strand of DNA to which a complementary nucleic acid strand can hybridize. A ligand may be an antibody molecule to which the corresponding antigen (epitope) can bind. A ligand may also include a particle on whose surface are a plurality of molecules to which other molecules may react. By using various markers and indicator molecules (e.g.: fluorescent dye molecules), the reaction between ligands and other molecules can be monitored and quantified. Thus, an array of different ligands immobilized on a biochip enables the-reaction and monitoring of multiple analyte molecules.
Many current passive biochip designs do not take full advantage of microfabrication and microelectronic technologies. Passive biochips cannot be readily used to achieve fully integration and miniaturization of the entire bioanalytical system from the front-end sample preparation to final molecular quantification/detection. In addition, passive biochips have other disadvantages including low analytical sensitivity, a long reaction time, and difficulties associated with control of temperature, pressure, and electrical fields at individual sites (called units) on the chip surfaces as well as difficulties in controlling the local concentrations of molecules.
On the other hand, active biochips allow versatile functions of molecular manipulation, interaction, hybridization reaction and separation (such as PCR and capillary electrophoresis) by external forces through means such as microfluidic manipulation and electrical manipulation of molecules. However, many such biochips cannot be readily used in high throughput applications. The electronic biochips developed by Nanogen can manipulate and control sample biomolecules with electrical field generated by microelectrodes, leading to significant improvement in reaction speed and detection sensitivity over passive biochips (see, U.S. Pat. Nos. 5,605,662, 5,632,957, and 5,849,486). However, to effectively move biomolecules in their suspension/solutions with electrical fields, the electrical conductivity of solutions has to be very low. This significantly limits the choice of buffer solutions used for biochemical assays because enzymes and other biomolecules are denatured under conditions of low ionic strength and/or serious non-specific binding occurs to chip surfaces.
The present invention provides a new type of active biochips in which magnetic forces are generated by individually addressable (controllable) units arranged in an array. The magnetic forces are used to control and manipulate magnetically modified molecules and particles and to promote molecular interactions and/or reactions on the surface of the chip. Magnetic forces have been widely employed in biological, biochemical and biomedical applications. For example, magnetic-activated cell sorting is a common technique based on selectively binding magnetic particles that has been modified with antibodies to specific cell types within a mixture. After binding, the cell-magnetic particle complexes from the cell mixture are selectively removed using a magnet. (See, for example, Miltenyi, S. et al. xe2x80x9cHigh gradient magnetic cell-separation with MACS.xe2x80x9d Cytometry 11:231-236 (1990)). Other examples were given in U.S. Pat. No. 5,439,586 describing a three-dimensional magnetic filter for separating magnetically labeled particles from non-magnetic particles in a flow stream and in U.S. Pat. No. 5,655,665 disclosing a micromachined magnetic particle separator for microfluidic magnetic separations.
The present invention discloses electromagnetic biochips that comprise individually addressable micro-electromagnetic units arranged in arrays. An array refers to a plurality of micro-electromagnetic units. An electromagnetic biochip may have single or multiple micro-electromagnetic unit arrays. Each unit is capable of inducing magnetic field upon the application of electric current, and is selectively addressable so that the magnetic filed generated by the unit can be turned on or off and/or can be modulated in terms of the field intensity and field direction through alteration of the electric current applied to the unit. Magnetic fields on the chip""s surface are then used to manipulate magnetic particles or magnetically-modified/loaded biomolecules. The magnetic particles or molecules are actually guided to predetermined locations on the chip""s surface. The chip""s surface may be chemically modified to form a functional layer for immobilizing ligand molecules so that affinity interaction or specific chemical reactions may occur between the ligand molecules and the magnetically guided particles or molecules. Magnetic guiding and manipulation of particles or molecules can increase the local concentration of these materials so as to increase the rate of biochemical or chemical reactions and the sensitivity of various assays. Because ionic strength and other buffer characteristics have little or no effect on magnetic fields, biochemically optimized buffer conditions can be selected. Furthermore, no strong electrical fields are present to complicate the assay or reaction by electrochemistry.
Micro-electromagnetic units are fabricated on substrate materials and generate individual magnetic fields when electric currents are applied. One example of the unit is a single loop of electrical conductor wrapped around a ferromagnetic body or core and connected to an electric current source through electronic switches. Such a loop may be a circle, ellipse, spiral, square, triangle or other shapes so long as a flow of electric current can be facilitated around the ferromagnetic body. If the loop is single, it should be complete or substantially complete. The loop may be in the form of a plurality of turns around the ferromagnetic body (either in one plane or stacked as in a coil). The turns may be fabricated within a single layer of the microstructure, or, alternatively, each turn may represent a separate layer of the structure. The electric conductor may be a deposited conductive tracexe2x80x94as in a electroplated, sputtered or deposited metallic structure, or the conductor may be formed within a semiconductor layer through selective doping. A preferred arrangement of an array of a plurality of micro-electromagnetic units has a column and row structure of the form common in microelectronics. That is, the columns and rows are mutually perpendicular although the columns and rows can readily be offset at different angles (e.g., 80 degrees).
The individual micro-electromagnetic units in a single chip may be of a single shape and dimension or there may be a variety of unit shapes and sizes within one chip. Characteristic dimensions of a unit vary from less than one micrometer to as large as one centimeter. The characteristic dimension refers to, for example, a diameter for a circle loop unit or a side length for a square loop unit. It will be apparent to one of ordinary skill in the art that where it is desired to react a large number of ligand molecules a larger unit size can be used. The units may be arranged in a regular, repetitive pattern (e.g., a rectangular grid) or they may be arranged in an xe2x80x9cirregularxe2x80x9d or xe2x80x9crandomxe2x80x9d pattern.
Individual micro-electromagnetic units may be selectively addressable so that at any instant there may be only a single energized unit generating a local magnetic field or there may be multiple energized units generating more or less complex magnetic fields. Addressing a micro-electromagnetic unit means applying electric current to energize the unit and to generate magnetic field in its vicinity. Electric current amplitudes and directions are selected so that energized units produce fields of sufficient intensity to attract and move magnetic particles or magnetically modified molecules. Units that are not selectively energized may be completely xe2x80x9coffxe2x80x9d (i.e., zero magnetic field) or such units may produce magnetic fields of insufficient intensity to attract or otherwise move the magnetic particles.
Selective addressing of individual units can be achieved in a number of ways. For example, where each unit contains a single loop of electric conductor one end of the loop can be connected to an electric current source (through electrical switching means) while the other end of the loops is attached to an electric current sink so that a current will flow through the loop. In another example, as explained below, units in a column/row array can be selectively activated by attaching (through switching means) a row to, for example, a current source and a column (through switching means) to a current sink. This will energize the unit at the intersection of the column and row.
The present invention further discloses methods for manipulating magnetic particles on electromagnetic chips. The particles may be suspended in a fluid (either aqueous or non-aqueous liquid or a gas) or even in a vacuum. When a micro-electromagnetic unit is energized, magnetic particles in the vicinity of that unit will experience magnetic forces and are attracted to the surface of the energized unit. That is, where a suspension of magnetic particles covers the entire chip array, energizing a single electromagnetic unit will affect only particles in the immediate vicinity of the energized unit. However, by sequentially energizing units it is possible to move and concentrate all of the magnetic particles suspended over the entire array. Such coordinated movement is referred to as xe2x80x9cmanipulationxe2x80x9d and such manipulation can be controlled by switching units on and off in a predetermined sequence. Manipulation of magnetic particles also refers to the change and control of particle position, velocity and other kinetic properties by modulating electric currents applied to micro-electromagnetic units and accordingly altering magnetic field distribution and forces acting on particles. Depending on the application, all units or some of the units may be energized simultaneously. Alternatively, units may be energized one-at-a-time.
Magnetic particles or materials used with the present invention may be of different sizes ranging from nanometer dimensions to micrometer or even millimeter dimensions. Magnetic particles may be of a variety of materials and be manufactured by a number of different processes as long as the magnetic fields produced by the biochips of the present invention can induce a sufficient magnetic dipole-moment in the particles.
The present invention further discloses methods for manipulating biomolecules/bioparticles, chemical-reagent molecules, drug molecules or any other molecules or particles with an electromagnetic biochip. These biochips can generally be used to manipulate any kind of magnetic particle. For controlling and handling non-magnetic particles and/or biomolecules, these materials are first magnetically modified. For example, the molecules may be covalently attached or physically absorbed to the surface of magnetic particles. The biomolecules may be proteins (e.g., antibodies, antigens and receptors), nucleic acids (e.g., single stranded DNA or RNA) or other molecules such as lipids or carbohydrates. The electromagnetic biochip surface may be modified for immobilizing ligand molecules that are capable of interacting with molecules on the surface of the manipulated magnetic particles. Such interactions are facilitated because the magnetic particles are concentrated at specific locations on which the appropriate ligand molecules are already immobilized.
In solutions, binding or reaction between molecules (e.g., antibody+antigen; specific DNA probe and its complementary single-stranded target DNA) occur as the molecules collide during diffusion. The efficiency and speed of the reactions depend on the local concentration of the reacting molecules and the kinetic energy of their collisions. In many biochip-based systems one type of molecule is immobilized at the chip surface while another type of molecule is present in a solution on the chip surface. Reactions occur when molecules passively diffusing in the solution collide with the immobilized molecules. Only a small percentage of the molecules in the solution actually diffuse and collide in a reasonable amount of time. Thus, the reactions are slow and inefficient, severely limiting the speed, efficiency and the sensitivity of bio/chemical assays performed on these biochips. In the electromagnetic biochips of the present invention the molecules in solution are actively brought into contact with the immobilized molecules on the chip surface by means of magnetic forces. The resulting reactions are xe2x80x9cactivelyxe2x80x9d driven by magnetic force leading to improved speed, efficiency and sensitivity.
For a typical magnetic particle of super-paramagnetic material, a magnetic dipole {overscore (xcexc)} is induced in the particle when it interacts with a magnetic field {overscore (B)}.                                                                                           μ                  _                                =                                                                            V                      p                                        ⁡                                          (                                                                        χ                          p                                                -                                                  χ                          m                                                                    )                                                        ⁢                                                            B                      _                                                              μ                      m                                                                                  ,                                                                          =                                                                    V                    p                                    ⁡                                      (                                                                  χ                        p                                            -                                              χ                        m                                                              )                                                  ⁢                                                      H                    _                                    m                                                                                        (        1        )            
where Vp is the particle volume, "khgr"p and "khgr"m are the volume susceptibility of the particle and its surrounding medium xcexcm is the magnetic permeability of medium, {overscore (B)} is the magnetic field strength. The magnetic force {overscore (F)}magnetic acting on the particle is determined by the magnetic dipole moment and the magnetic field gradient:
{overscore (F)}magnetic=xe2x88x920.5Vp("khgr"pxe2x88x92"khgr"m){right arrow over (H)}m∘∇{right arrow over (B)}m,xe2x80x83xe2x80x83(2)
where the symbols xe2x80x9c∘xe2x80x9d and xe2x80x9c∇xe2x80x9d refer to dot-product and gradient operations, respectively. The particle velocity xcexdparticle under the balance between magnetic force and viscous drag is given by:                               v          particle                =                                            F              _                        magnetic                                6            ⁢            π            ⁢                          xe2x80x83                        ⁢            r            ⁢                          xe2x80x83                        ⁢                          η              m                                                          (        3        )            
where r is the particle radius and xcex7m is the viscosity of the surrounding medium. Thus to achieve sufficiently large magnetic manipulation force, the following factors should be considered:
(1) Particle susceptibility should be maximized;
(2) Magnetic field strength should be maximized; and
(3) Magnetic field strength gradient should be maximized.
We will now describe several illustrative embodiments of the present invention. According to one embodiment of the present invention, an individual addressable micro-electromagnetic unit column-row array chip comprises:
a substrate;
an array of cavities on the substrate;
a ferromagnetic core in each cavity;
a first layer of conductive traces on the substrate running between the columns of ferromagnetic cores;
a first insulation layer on the substrate surface that covers the first layer of conductive traces;
a second layer of conductive traces on the surface of the first insulation running between the rows of ferromagnetic cores, perpendicular to the first conductive traces;
a second insulation layer on the chip surface that covers the ferromagnetic core array and the second layer of conductive traces.
In another embodiment of the present invention, an electromagnetic biochip comprises an individually addressable micro-electromagnetic unit array chip that comprises
a substrate;
an array of cavities on the substrate;
an magnetic-core in each cavity;
a first layer of conductive traces on the substrate running between every columns of magnetic-cores;
a first insulation layer on the substrate surface that covers the first layer of conductive traces;
a second layer of conductive traces on the surface of the first insulation layer running between each rows of magnetic-cores perpendicular the first layer conductive traces;
a second insulation layer on the chip surface that covers the magnetic-core array and the second layer of conductive traces;
a thin binding layer (i.e., a functional layer) that covers the second insulation layer and is used to immobilize ligand molecules thereon; and
ligand molecules that are directed and immobilized onto the thin functional layer using magnetic forces or other methods.
The functional layer is used for immobilizing ligand molecules. Examples of a functional layer include, but are not limited to, a molecular monolayer, a membrane, a gel, and a porous or non-porous material layer. The functional layer may be an additional layer adhered to the biochip surface (in the above example, to the second insulation layer). Alternatively, the functional layer may be formed by direct chemical-modification of the biochip surface molecules. In the example above, the surfaces of the second insulation layer may be chemically modified to contain chemical groups or molecular sites for binding or attaching ligand molecules. Ideally, the functional layer should show minimal or no non-specific bindings to molecules other than ligand molecules and should allow efficient binding or attachment of ligand molecules.
According to one embodiment of the present invention, a method for manipulating biomolecules, chemical reagents, or drug molecules comprises these steps:
providing the above-described individually addressable micro-electromagnetic unit array chips;
forming a thin binding layer (i.e., a functional layer) for immobilizing ligand molecules on the chip""s surface;
loading micro-locations on the binding layer with a set of ligand molecules by positioning and immobilizing magnetically-modified ligand molecules at predetermined micro-locations to form molecule-binding regions on the chip surface by selectively controlling electric current in the conductive traces in the micro-electromagnetic unit array chip to produce magnetic fields around desired micro-electromagnetic units;
magnetically modifying or loading target molecules by linking them with magnetic beads;
introducing solutions containing magnetic bead-linked target molecules onto the above-described ligands-containing micro-electromagnetic unit array chip;
producing magnetic fields around desired micro-locations by selectively addressing and energizing particular units within micro-electromagnetic unit array so that magnetically modified target molecules can be directed toward ligand molecules on the desired unit locations so as to accomplish binding reactions; and
releasing magnetic beads from target molecules followed by removal of the magnetic beads.
The ligands and target molecules in the above method may be biological molecules, chemical reagents, drug-candidate molecules, or any other molecules or particles. Methods according to the present invention may be used for hybridization and detection for specific sequences of DNA molecules, for antibody/antigen binding interaction in application areas such as drug screening, bio/chemical (i.e., biochemical or chemical) process control, biochemical monitoring and clinical diagnosis.
In the, following, with the aid of figures wherein like structures are denoted by like reference signs, we provide detailed descriptions of exemplary embodiments of individually addressable electromagnetic array chips, electromagnetic biochips, and methods of manipulating molecules.